Development and Evaluation of Event-Specific Qualitative PCR Methods for Genetically Modified Bt10 Maize

Abstract

In 2005 it was reported that the genetically modified (GM) maize strain or "event" called Bt10 had been distributed inadvertently in the United States over the previous 4 years. In order to ensure that grain for food and feed production did not contain trace amounts of Bt10 maize and complied with the applicable regulation, highly sensitive and specific detection of Bt10 maize was required. Accordingly, we developed a novel qualitative PCR system for specific detection of Bt10 maize. Moreover, we amply evaluated the performance characteristics of two PCR systems, our own and the one provided by the developer of Bt10, Syngenta Co. Ltd. It was confirmed that both of the qualitative PCR systems can specifically detect Bt10 maize, and the results of a single-laboratory examination suggested that the limit of detection was approximately less than 0.05% for both methods. To evaluate the reproducibility of the methods, we organized an interlaboratory study with the participation of 6 laboratories and analysis of 240 blind test samples. In this paper, we report, for the first time, the statistical analysis of the qualitative PCR data obtained from the interlaboratory study. The results of this analysis also revealed that there was no significant difference in the sensitivity between the two aforementioned methods and that the limit of detection of both the methods was less than 0.05%. Thus, we conclude that both of the methods are equally suitable for correct identification and sensitive detection of the unapproved GM maize Bt10 event in test samples.

A Screening Method for the Detection of the 35S Promoter and the Nopaline Synthase Terminator in Genetically Modified Organisms in a Real-Time Multiplex Polymerase Chain Reaction Using High-Resolution Melting-Curve Analysis

“…Previous studies have demonstrated the detection of GM crops using specific probes. 7,13) However, the use of fluorescence labeled probes adds significant cost to an assay comprising multiple targets. The closed-tube screening method using HRM has advantages over current techniques because it requires no post-PCR handling, which minimize the risk of PCR contamination, and no separation step, both of which improve analysis time.…”

“…Previously, we reported the development of qualitative detection methods for GM maize, GM potatoes (NewLeaf Plus, NewLeaf Y), GM papayas (Line 55-1 or its derivatives) and GM rice (LL rice and Chinese Bt rice lines), including qualitative polymerase chain reaction (PCR) methods and a histochemical assay. [4][5][6][7][8][9][10][11][12][13] Especially, in China, some GM rice varieties have been developed and tested in the field and environmental trials. [14][15][16] Bt rice expressing the Bt toxin has been developed in China and approved for environmental release trials.…”

Development of Multiplex PCR Method for Simultaneous Detection of Four Events of Genetically Modified Maize: DAS-59122-7, MIR604, MON863 and MON88017

“…Seeds of GM maize, i.e., DAS-59122-7, MON863 and MON88017, and combined event products of GM maize were ground with a Multibeads Shocker (Yasui Kikai Co., Osaka, Japan) on a single-seed basis, and DNA of each kernel was extracted with the DNeasy Plant Mini kit (QIAGEN GmbH, Hilden, Germany) following the standard experimental procedure for GM analysis in foodstu#s in Japan῎ 6 . Other maize and other plant seed materials were ground with a P-14 seed rotor mill (Fritsch GmbH, Ibar-Oberstein, Germany), and used for DNA extraction.…”

Interlaboratory Validation of an Event-Specific Real Time Polymerase Chain Reaction Detection Method for Genetically Modified DAS59132 Maize

“…Genomic DNAs were extracted from the flour samples using a silica-gel membrane-type kit (DNeasy Plant Mini; QIAGEN, Hilden, Germany) with modifications to the kit manual as described previously 7), 8), 15) . The DNA concentration was determined by measuring the UV absorption at 260 nm using an ND-1000 spectrophotometer (NanoDrop Technologies Inc., Rockland, DE).…”